Replication of Human Endogenous Retroviruses in Modern Humans

描述（由申请人提供）：不编码人类细胞蛋白的约98%的人类基因组的功能仍有待阐明。活跃复制的内源性逆转录病毒在数百万年前进入人类基因组，并成为遗传物质的稳定部分，逆转录病毒元件目前占现代人类基因组的约8%。这些病毒随后获得了多个突变，导致人们普遍假设它们不再能够复制。然而，在研究活体患者而不是标准细胞系时，我们最近发现了令人惊讶的证据，表明在某些癌症患者中，HERV-K（HML-2），一种内源性逆转录病毒，是一种相对较新的进入人类基因组并与肿瘤发生有关的病毒，可能仍然能够复制。然而，内源性逆转录病毒的复制和传播很难使用标准技术来证明，因为这些病毒已经存在于所有人类细胞的基因组中。因此，我们组建了一个多元化的专家研究小组，他们将测试现代HERV-K（HML-2）可以使用尖端和互补技术复制的假设。我们将使用一种新设计的分子系统，其中抗生素耐药性作为替代标记，以评估我们是否可以在实验室中从患者血液中传代病毒。我们还将使用高通量DNA测序和生物信息学来寻找存在于癌症患者中但未在当前人类基因组草案中表示的全长功能性病毒。将使用从生物信息学研究中获得的信息和/或通过从患者血液中发现的HERV-K RNA克隆全长病毒，制备代表现代HERV-K（HML-2）的感染性克隆。然后将该克隆用绿色荧光蛋白标记并用于直接研究复制。基于我们使用核磁共振（NMR）可视化指导RNA和蛋白质构象转变的空间相关动力学的进展，我们还将开发NMR方法来可视化HERV-K（HML-2）体内复制。内源性逆转录病毒仍然可以在现代人类中复制的证据将导致对这些病毒的思考范式转变，并将表明它们在重塑个体基因组中的作用。此外，由于染色体内源性逆转录病毒序列的表达增加与癌症和自身免疫有关，这些发现将有助于理解重大疾病的发病机制。最后，如果能够在特定个体的血液中发现能够引起疾病的复制性传染性内源性人类逆转录病毒，则该信息将对血液供应的安全性具有重要意义。

DESCRIPTION (provided by applicant): The functions of the approximately 98% of the human genome that do not encode human cellular proteins remain to be elucidated. Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material, with retroviral elements presently making up approximately 8% of the modern human genome. These viruses subsequently acquired multiple mutations, leading to the widely-held assumption that they are no longer competent to replicate. However, in studying living patients rather than the standard cell lines, we have recently discovered surprising evidence suggesting that in certain patients with cancer HERV-K (HML-2), an endogenous retrovirus that is a relatively recent entrant into the human genome and has been linked to oncogenesis, might still be capable of replication. Replication and transmission of endogenous retroviruses is difficult to prove using standard techniques, however, as these viruses are already present in the genomes of all human cells. Therefore, we have assembled a diverse and expert group of investigators who will test the hypothesis that modern HERV-K (HML-2) can replicate using cutting-edge and complementary techniques. We will use a newly devised molecular system in which antibiotic resistance serves as a surrogate marker to assess whether we can passage virus from the blood of patients in the laboratory. We will also use high-throughput DNA sequencing and Bioinformatics to find full-length functional virus that is present in cancer patients but is not represented in the current draft of the human genome. An infectious clone that is representative of modern HERV-K (HML-2) will be made by using the information gained from the Bioinformatics studies and/or by cloning a full-length virus from the HERV-K RNA found in the blood of patients. This clone will then be tagged with green fluorescent protein and used to directly study replication. Building on our advances using Nuclear Magnetic Resonance (NMR) to visualize spatially correlated dynamics that direct RNA and protein conformational transitions, we will also develop NMR methods to visualize HERV-K (HML-2) replication in vivo. Proof that endogenous retroviruses can still replicate in modern humans will lead to a paradigm shift in thinking about these viruses, and will suggest a role for them in reshaping individual genomes. In addition, as increased expression of chromosomal endogenous retroviral sequences has been linked to cancer and autoimmunity, these findings will be relevant to understanding the pathogenesis of significant diseases. Finally, if replicating, infectious endogenous human retroviruses capable of causing disease can be found in the blood of given individuals, this information will have important implications for the safety of the blood supply.